CN102453074A - 五环三萜类皂苷及其用途 - Google Patents
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Abstract
本发明涉及医药技术领域，是一类从毛茛科植物朝鲜白头翁(Pulsatilla cernua(Thunb.)Bercht.et Opiz.)中分离得到的五环三萜类化合物，其化学结构式如图。其中glc、rha和ara分别代表β-D-吡喃葡萄糖、α-L-吡喃鼠李糖和α-L-吡喃阿拉伯糖。化合物A、B均为新化合物。体外细胞试验表明，该化合物对Aβ(25～35)诱导的神经母细胞瘤细胞株SH-SY5Y损伤具有明显的保护作用，本发明化合物制备方法简单，活性显著，本发明为预防和治疗老年性痴呆(阿尔茨海默病，Alzheimer’s disease，AD)提供了新化合物。
Description
技术领域
本发明涉及医药技术领域，涉及五环三萜类皂苷及其用途，具体涉及一种从毛茛科植物朝鲜白头翁(Pulsatilla cernua(Thunb.)Bercht.et Opiz.)中分离得到的五环三萜类化合物及其在预防和治疗老年性痴呆(阿尔茨海默病，Alzheimer’s disease，AD)药物中的应用。
背景技术
阿尔茨海默病(Alzheimer’s disease，AD)由神经病理学家阿尔茨海默于1906年首先发现，并以其名字命名。AD是一种弥漫性中枢神经退化性疾病，以进行性认知障碍，智力衰退和人格改变为特征的常见老年性神经变性疾病，约占所有痴呆的50％～60％。据报道，65～74岁的老年人中约3％患有AD，75～84岁的人群中患病率为19％，而＞85岁的老年人中AD患者则高于47％，已成为老年人的第四大死因。虽然发展中国家缺少权威性和代表性的数字统计，但估计世界上60％的痴呆患者存在于发展中国家。随着世界人口老龄化的进展，这一问题将更加突出。因此，有关该病的治疗研究刻不容缓。
AD的病理变化主要发生于前脑基底、海马和大脑皮质，其主要病理特征为：1)颞叶和海马皮质等部位神经元丢失；2)神经原纤维缠结(neurofibrillary tangles，NFTs)；3)老年斑(senileplaques，SPs)，其主要成分为β淀粉样蛋白(β-amyloid peptide，Aβ)；4)脑血管淀粉样病变。目前普遍认同的AD主要发病机制：具有神经毒性的Aβ在脑实质沉积，启动病理级联反应，形成NFT，导致广泛的神经元丢失。脑组织的破坏导致功能的损害，出现痴呆症状。Aβ沉积所形成的SP是AD的主要病理学特征。
朝鲜白头翁(Pulsatilla cernua(Thunb.)Bercht.et Opiz.)为毛茛科(Ranunculaceae)植物，主要分布于我国东北地区。具有清热解毒、凉血止痢的功效。其中三萜皂苷类为该植物的主要化学成分。本发明中的化合物为新化合物，未见相关报道。
发明内容
本发明提供一类从朝鲜白头翁中分离得到具有预防和治疗老年性痴呆(阿尔茨海默病，Alzheimer’s disease)活性的五环三萜类化合物，其化学结构式如下：
其中，R1、R2及R3分别为：
R1 R2 R3
A CH3 S4 S1
B CHO S3 S2
S1＝-glc(6→1)glc(4→1)rha
S2＝-g lc(6→1)glc
S3＝-ara[(4→1)glc](2→1)rha(3→1)glc
S4＝-ara[(4→1)glc](2→1)rha(3→1)glc(4→1)glc
化合物A、B均为新化合物。glc、rha和ara分别代表β-D-吡喃葡萄糖、α-L-吡喃鼠李糖和α-L-吡喃阿拉伯糖。
本发明化合物制备方法如下：
1)提取：将朝鲜白头翁干燥根粉碎，提取；
2)浓缩：提取液浓缩或减压浓缩得浓缩液；
3)萃取：将浓缩液以溶剂萃取得到萃取液；
4)浓缩：将3)中所得正丁醇萃取液浓缩或减压浓缩；
5)纯化：步骤4)所得浓缩液经大孔吸附树脂柱色谱，梯度洗脱。洗脱液减压浓缩、干燥，即得朝鲜白头翁总皂苷。将朝鲜白头翁总皂苷通过反复硅胶柱层析，并以制备HPLC分离纯化，得到化合物。
其中，
步骤1)所述提取方法是以50％乙醇水溶液回流提取。
步骤3)所述萃取所用溶剂依次为石油醚、二氯甲烷及正丁醇，各萃取次数2次，溶剂用量每100g药材100mL溶剂。
步骤4)所述萃取液为正丁醇萃取液。萃取次数2次，溶剂用量每100g药材100mL溶剂。
步骤5)所述大孔吸附树脂为D101型大孔吸附树脂。洗脱溶剂依次为水、30％乙醇、50％乙醇、70％乙醇和95％乙醇。
本发明所述的五环三萜类化合物对Aβ(25～35)诱导的神经母细胞瘤细胞株SH-SY5Y损伤具有明显的保护作用，具有预防和治疗老年性痴呆的作用。
附图说明
图1为化合物A的主要HMBC相关
图2为不同剂量的化合物A处理18h后SH-SY5Y细胞株受Aβ(25-35)的影响与空白组(#p＜0.05)和模型组(*p＜0.05)的比较
图3为不同剂量的化合物B处理18h后SH-SY5Y细胞株受Aβ(25-35)的影响与空白组(##p＜0.01)和模型组(*p＜0.05，**p＜0.01)的比较
具体实施方式
实施例1.化合物制备
将朝鲜白头翁干燥根(100g)粉碎，以50％乙醇水溶液于80℃下回流提取4h，提取液减压浓缩得浓缩液，浓缩液依次用100mL石油醚、二氯甲烷及正丁醇萃取，分别萃取2次，正丁醇萃取液浓缩后经D101大孔吸附树脂柱色谱，依次以水、30％乙醇、50％乙醇、70％乙醇和95％乙醇梯度洗脱。合并50％和70％乙醇洗脱液，减压浓缩、干燥，经硅胶柱层析，以二氯甲烷-甲醇-水(9∶1∶0.1～7∶3∶0.3)洗脱，再经制备HPLC分离纯化，以甲醇-乙腈-水(3∶2∶3)为流动相洗脱，得到化合物。
结构鉴定
化合物A，白色无定形粉末(甲醇)，Liebermann-Burchard和Molish反应均为阳性。m.p.270～273℃。ESI-MS：m/z 1690.1[M-H]-，1715.2[M+Na]+，HR-TOF-MS显示[M-H]-准分子离子峰(m/z 1689.7773，计算值1689.7752)，分子式为C77H126O40。1H NMR(600MHz，pyridine-d5)高场给出7个单峰甲基特征信号，分别为δ0.82，0.84，0.84，1.03，1.12，1.19，1.26。两个双峰甲基信号δ1.52(3H，d，J＝6.0Hz)，1.66(3H，d，J＝6.0Hz)，低场区给出一个烯氢信号δ5.35(1H，t-like)以及八个糖端基氢信号δ4.61(1H，overlapped)，4.96(1H，d，J＝7.2Hz)，5.10(1H，d，J＝7.8Hz)5.15(1H，d，J＝7.8Hz)，5.42(1H，d，J＝7.8Hz)，5.84(1H，brs)，6.19(1H，brs)，6.22(1H，d，J＝7.8Hz)。13C NMR(150MHz，pyridine-d5)低场区δ176.5(酯基碳)，144.2和122.9(烯碳)。以上信息提示该化合物可能为Δ12-齐墩果烷型皂苷。与齐墩果酸的13C NMR谱数据比较，苷元部分C-3向低场位移，C-28向高场位移。在δ90～110区间给出八个糖端基碳信号δ95.4，101.3，102.5，104.6，104.8，105.1，106.3，106.4。推断该化合物为苷元的C-3及C-28发生苷化的双糖链皂苷。通过HSQC对苷元及糖的部分信号进行归属。HMBC显示，δ4.61(1H，overlapped，Ara H-1)与δ88.4(C-3)相关，δ5.10(1H，d，J＝7.8Hz，Glc H-1)与δ79.9(Ara-4)，δ6.19(1H，brs，Rha H-1)与δ75.7(Ara-2)，δ5.42(1H，d，J＝7.8Hz，Glc’H-1)与δ83.0(Rha-3)，δ5.15(1H，d，J＝7.8Hz，Glc””H-1)与δ80.9(Glc’-4)分别相关(见图1)。确定苷元C-3位糖链的连接顺序为-β-D-吡喃葡萄糖基(1→4)-β-D-吡喃葡萄糖基(1→3)-α-L-吡喃鼠李糖基(1→2)[β-D-吡喃葡萄糖基(1→4)]-α-L-吡喃阿拉伯糖基。δ6.22(1H，d，J＝7.8Hz，Glc”H-1)与δ176.5(C-28)相关，4.96(1H，d，J＝7.2Hz，Glc”’H-1)与δ68.9(Glc”-6)，5.84(1H，brs，Rha’H-1)与δ77.9(Glc”’-4)。确定C-28位糖链顺序为-α-L-吡喃鼠李糖基(1→4)-β-D-吡喃葡萄糖基(1→6)-β-D-吡喃葡萄糖基。进而确定化合物结构为齐墩果酸3-O-β-D-吡喃葡萄糖基(1→4)-β-D-吡喃葡萄糖基(1→3)-α-L-吡喃鼠李糖基(1→2)[β-D-吡喃葡萄糖基(1→4)]-α-L-吡喃阿拉伯糖基-28-O-α-L-吡喃鼠李糖基(1→4)-β-D-吡喃葡萄糖基(1→6)-β-D-吡喃葡萄糖酯苷(oleanolic acid3-O-β-D-glucopyranosyl(1→4)-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester)。该化合物未见文献报道。其13C NMR数据见表1～2。
化合物B，白色无定形粉末(甲醇)，Liebermann-Burchard和Molish反应均为阳性。HR-TOF-MS显示[M-H]-准分子离子峰(m/z 1541.6973，计算值1541.7017)，分子式为C71H114O36。1H NMR(600MHz，pyridine-d5)高场给出6个单峰甲基特征信号，分别为δ0.79，0.81，0.81，0.95，1.13，1.39。两个双峰甲基信号δ1.50(3H，d，J＝6.0Hz)，1.60(3H，d，J＝6.0Hz)，低场区给出一个烯氢信号δ5.30(1H，t-like)以及七个糖端基氢信号δ4.61(1H，overlapped)，4.90(1H，d，J＝7.8Hz)，5.11(1H，d，J＝7.8Hz)5.55(1H，d，J＝7.8Hz)，5.76(1H，brs)，6.12(1H，brs)，6.14(1H，d，J＝8.4Hz)。13C NMR(150MHz，pyridine-d5)低场区δ205.5(醛基碳)，δ176.5(酯基碳)，144.2和122.9(烯碳)。以上信息提示该化合物可能为Δ12-齐墩果烷型皂苷，且苷元为丝石竹苷元(gypsogenin)。苷元部分C-3向低场位移，C-28向高场位移。在δ90～110区间给出七个糖端基碳信号δ95.8，101.3，102.9，103.1，105.0，105.2，106.7。推断该化合物为苷元的C-3及C-28发生苷化的双糖链皂苷。通过对照化合物A的糖部分NMR数据，确定化合物结构为丝石竹苷元3-O-β-D-吡喃葡萄糖基(1→3)-α-L-吡喃鼠李糖基(1→2)[β-D-吡喃葡萄糖基(1→4)]-α-L-吡喃阿拉伯糖基-28-O-α-L-吡喃鼠李糖基(1→4)-β-D-吡喃葡萄糖基(1→6)-β-D-吡喃葡萄糖酯苷(gypsogenin3-O-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester)。该化合物未见文献报道。其13C NMR数据见表1～2。
表1化合物A、B的13C NMR数据(苷元部分，pyridine-d5)
表2化合物A、B的13C NMR数据(糖部分，pyridine-d5)
体外活性试验
人神经母细胞瘤细胞SH-SY5Y生长于MEM/F12+10％胎牛血清培养液中，贴壁生长，培养在37℃，含有5％CO2的培养箱里。SH-SY5Y细胞以8000个/孔的密度接种在96孔板中，培养24小时后加入不同浓度的样品，作用18小时后加入Aβ(25-35)20μmol/L，孵育30小时后，向各孔中加入5mg/mL MTT 15μL，放入二氧化碳培养箱孵育4小时，4小时后将各孔中液体吸出，加入DMSO150μL，振摇10分钟后于酶标仪492nm处测定吸光度值，计算细胞存活率。
表3MTT法测定化合物活性结果(μmol/L)
由表3及图2～3可见，化合物对Aβ(25～35)诱导的神经母细胞瘤细胞株SH-SY5Y损伤具有明显的保护作用，具有预防和治疗老年性痴呆(阿尔茨海默病，Alzheimer’s disease)的作用。
Claims (6)
1.五环三萜类皂苷，其特征在于化学结构式如下：
glc、rha和ara分别代表β-D-吡喃葡萄糖、α-L-吡喃鼠李糖和α-L-吡喃阿拉伯糖。
2.如权利要求1所述的五环三萜类皂苷的制备方法，其特征在于，步骤如下：
1)提取：将朝鲜白头翁干燥根粉碎，加入溶剂提取；
2)浓缩：提取液浓缩或减压浓缩得浓缩液；
3)萃取：将浓缩液以溶剂石油醚、二氯甲烷及正丁醇萃取得到萃取液；
4)浓缩：将3)中所得正丁醇萃取液浓缩或减压浓缩；
5)纯化：步骤4)所得浓缩液经大孔吸附树脂柱色谱，梯度洗脱，洗脱液减压浓缩、干燥，即得朝鲜白头翁总皂苷；将朝鲜白头翁总皂苷通过反复硅胶柱层析，并以制备HPLC分离纯化，得到化合物。
3.根据权利要求2所述的五环三萜类皂苷的制备方法，其特征在于，步骤1)的提取方法为热回流提取法，所用溶剂为50％乙醇水溶液，提取温度为80℃。
4.根据权利要求2所述的五环三萜类皂苷的制备方法，其特征在于，所述大孔树脂为D101型大孔树脂，梯度洗脱所用溶剂依次为水、30％乙醇、50％乙醇、70％乙醇和95％乙醇。
5.根据权利要求2所述的五环三萜类皂苷的制备方法，其特征在于，所述萃取溶剂的用量为每100g药材100mL，提取次数2次。
6.权利要求1所述的五环三萜类皂苷在制备预防和治疗老年性痴呆药物中的应用。
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| CN113633682A (zh) * | 2021-07-22 | 2021-11-12 | 广西馨海药业科技有限公司 | 一种治疗带状疱疹的乳膏剂及其制备方法 |
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| CN103356656B (zh) * | 2013-07-04 | 2015-11-25 | 顾祥茂 | Chukrasone A在制备治疗阿尔茨海默病药物中的应用 |
| CN113633682A (zh) * | 2021-07-22 | 2021-11-12 | 广西馨海药业科技有限公司 | 一种治疗带状疱疹的乳膏剂及其制备方法 |
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